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Summer Research Fellowship Programme of India's Science Academies

Role of frequently found p53 Gain-of-function mutations in ovarian carcinoma in inducing stemness

Bidisha Dey

B.tech-M.tech Dual Degree in Biotechnology, KIIT School of Biotechnology, KIIT (Deemed to be University), Bhubaneswar

Prof. Susanta Roychoudhury

Chief, Basic Research, SGCCRI, Thakurpukur, Kolkata

Abstract

Globocan 2018 (International Agency for Research on Cancer) has reported ovarian cancer as the 3rd leading cancer among Indian women with the incidence and mortality of 36,170 and 24,015, respectively. High-Grade Serous ovarian cancer (HGSOC) is the most frequent ovarian Epithelial cancer (Type II), which features aggressiveness, chemoresistance within 6-12 months and low survival. More than 96% cases are seen to have p53 mutations. According to The Cancer Genome Atlas, R175H and R273H are two of the most frequent p53 missense mutations in HGSOC. These mutations are responsible for Gain Of oncogenic Functions (GOF) in the mutant p53 added to the loss of its tumor suppressor ability. Oncogenic functions of GOF p53 mutants include proliferation, migration, Epithelial-to-Mesenchymal Transition (EMT), chemoresistance. Previous research works in other types of cancers have shown that chemoresistance is associated with cancer stem cells. We aim to find out whether these GOF p53 mutations are responsible for stemness in characteristically chemoresistant HGSOC. We are simultaneously performing our study on clinical samples as well as on HGSOC cell lines. We checked for the presence of p53 mutations by performing Immunohistochemistry (IHC) in clinical samples. On the other hand, we used p53 null cell lines (SKOV-3) to transfect mutant p53 plasmid and check its expression by Western Blot. We compared the p53 expression of SKOV-3 with 5-FU treated wild type A2780 cell line (drug treatment stabilizes wild type p53, which otherwise undergoes MDM 2 mediated proteasomal degradation). We are in the process of making stable cell line expressing the p53 mutants. We have successfully prepared blocks of 10 clinical tissue samples, for further H & E staining and IHC which is in the process of standardization. Also we will be analysing the transcript-level and protein-level expressions of stemness markers by different molecular methods.

 Keywords: TP53, cancer cell line, HGSOC, GOF

 Abbreviations

Abbreviations
HGSOC High Grade Serous Ovarian Cancer
LGSOC Low Grade Serous Ovarian Cancer
GOF Gain Of Function
CSC Cancer Stem Cells
DBD DNA Binding Domain
IHC Immunohistochemistry

INTRODUCTION

Ovarian Cancer

Ovarian cancer is one of the most lethal gynecological conditions. Women in the older age group, with inherited BRCA mutations, those involved in Estrogen Replacement Therapy, those early onset of menstruation and late menopause, are at a higher risk for developing ovarian cancers (Jayson et al 2014). Being the 3rd commonest type of gynecological cancers, ovarian cancers can be divided into epithelial ovarian cancer, germ cell tumors and sex cord tumors. Approximately 90% of the primary ovarian tumors are surface epithelial cancers which can further be divided into low grade serous (Type I) and high grade serous (Type II). High grade serous ovarian cancer is the most frequently occurring surface epithelial cancer which is characterized by aggressiveness, chemoresistance, difficultly in early detection etc. HGSOCs are detected mostly after stage II [FIGO], so range of effective treatment options, narrow down (Marks et al 1991). These Type I and Type II Surface Epithelial cancers are associated with various types of mutation. Most LGSOCs involves mutation in PTEN, KRAS, PIK3C genes. LGSOCs are and less aggressive than HGSOCs, making HGSOCs a matter of concern.

More than 96% cases of HGSOCs are associated with p53 mutations p53 mutations are mainly missense mutations which involves change in one amino acid to other (Brachova et al 2013). According to The Cancer Genome Atlas (TCGA), there are 6 hotspot mutations occuring frequently among which mutations R175H and R273H are two of the most frequent p53 missense mutations in HGSOC. These mutations belong to the group of Gain-of-Function mutations that are characterized by loss of tumor suppressor ability of p53 and gain of oncogenic functions like chemoresistance, migration, Epithelial-to-Mesenchymal Transition (EMT), proliferative properties, etc. (Shetzer et al 2014 ). 

High Grade Serous Ovarian Cancer and Associated Problems of Chemoresistance

According to GLOBOCAN report 2018, ovarian cancer has a high incidence rate which numbers to 36,170 and a death of 24,015 every year in India. The five year survival rate when compared to LGSOC is also extremely low. Due to lack of appropriate symptoms in patients and unavailability of proper screening methods, specially in developing countries like India, HGSOCs become extremely difficult to be detected and diagnosed. Existing screening methods of ovarian cancer like CA125 are of great prominence. HGSOCs when detected can be treated directly by performing surgery, which is also used in debulking and staging of the tumor.

Surgery solely is a good option when the cancer is confined to the ovaries, but when metastasis occur, as we know HGSOCs are highly aggressive, surgery along with Adjuvant or Neo-adjuvant Chemotherapy (NACT) is preferred. Platinum based chemotherapy which involves. Carboplatin, cisplatin and Taxane based chemotherapy which involves treatment with Paclitaxal are highly in use, in most cases of cancers. Inspite of these chemotherapy options, one major hindrance that is faced during treatment for HGSOCs is chemoresistance and as a result recurrence of the cancer within a year. According to earlier research works, in other cancers a connection has been drawn out between chemoresistance and stemness in presence of GOF mutant p53 in cancer cells. We are trying to find out this relation in case of ovarian cancer as well, which could possibly be the reason for recurrence and extreme aggressiveness of HGSOCs.

Main Objective

“Role of frequently found p53 Gain-of-Function mutations in ovarian carcinoma, in inducing stemness.”

Sub-objectives

A) To check for the expression of p53 protein in p53 null cell line after transfecting the p53 mutant plasmid.

B) To check for cells exhibiting stem cell characters by performing side population assay.

C) To check for stemness markers in transiently transfected SKOV-3 cell lines with the help of Western Blot.

 D) To check for drug resistance in ovarian cancer cells by performing MTT assay.

Scope

From this project we expect to see the relation of stemness and chemoresistance in ovarian cancer cells possessing p53 Gain-of-Function mutations like R175H and R273H. The relation between p53 GOF mutation and stemness, also should be well understood to find out solution of aggressive spreading of HGSOC.

LITERATURE REVIEW

A) p53- p53 is a tumor suppressor protein, encoded by TP53 gene which regulates cell cycle to suppress tumor formation, hence resists cancer. TP53 gene is located in the chromosome 17 and yields the 393 amino acid containing p53. Being a homotetrameric protein, p53 contains 4 domains that each have different functions. First, the acidic N terminal transcriptional activation domain (TAD) which activates transcription factors. Second, the central DNA binding core domain (DBD) which binds to typical responsive elements in the DNA. Third, the C terminal tetramerization domain and lastly the domain that recognises DNA damage (Sabapathy & Lane 2018). In stressed conditions the cellular levels of p53 due to post-translational mechanisms, increase which leads to cell cycle arrest or apoptosis. Under non-stressed conditions, p53 is controlled by the action of E3 ligase MDM2 mediated proteasomal degradation. In case of ovarian cancer, missense mutations like R273H, R175H, R248Q confer oncomorphic activity to p53 protein (Vassilev et al 20 2004). These oncomorphic activities can be explained by four distinct mechanisms: In the first mechanism, alteration of sequence specificity which are required for p53 transactivation of target genes due to particular missense mutation takes place. Secondly, oncomorphic p53 binds to non typical transcription factors which help in indirect transcriptional activation of gene. In the third mechanism, the oncomorphic p53 binds and sequesters other transcriptional factors necessary for normal stress response. In the fourth mechanism, oncomorphic p53 interacts with novel proteins to increase or decrease their activity (Brachova et al 2013).

B) R175H and R273H p53 mutation- According to TCGA data, R175H and R273H are two of the most frequent p53 mutations respectively in ovarian cancer. ‘R175H’ and ‘R273H’ are missense mutations in which Arginine is replaced by Histidine at the 175th and 273rd position respectively in the amino acid chain. p53 proteins mainly have 6 hotspot mutations including R175H and R273H which are found to affect the DNA- binding domain of the TP53 protein. (Oren & Rotter 2010).

C) Cancer stem cell- Stem cells are defined by the ability of self‐renewal, generation of differentiated cells of an organ. Stem cells are the only normal cells that share cancer cells' intrinsic self renewal ability. According to previous researches in ovarian cancer, a small, phenotypically distinct subset of the cancer cells (tumorigenic cells, or cancer stem cells) has the exclusive ability to form tumors.

The rest of the cells, which form the bulk of the tumor, are unable to self‐renew or sustain tumorigenesis. Cancer stem cells are associated with drug resistance which is achieved by implementation of various mechanisms.

D) Role of GOF mutant p53 in stemness in cancer- GOF mutant p53 performs multi-activity by which the cancer cells are rendered drug resistant. Efflux of drugs out of cells, downregulation of pro-apoptotic proteins, upregulation of anti-apoptotic proteins, enhanced expression of ABC transporters in the cell membrane etc. are some mechanisms that the mutant p53 performs. As a result of drug resistance, these cancer cells live on and start behaving like cancer stem cells (CSCs).

E) Yamanaka factors- Stem cell markers like Oct-4 and c-Myc (also called Yamanaka Factors) have the potential to induce pluripotency in both mouse and Human Somatic cells when over-expressed. This fact indicates that these factors regulate the developemental signalling network required for Embryonic Stemcell pluripotency (Zhao et al 2017). Klf-4, Sox-2, Oct-3 are other Yamanaka Factors.

F) Stem character was assessed by analyzing expression of Yamanaka factors and side population assay- Stem character is a consequence of p53 GOF mutation.Cancer cells start to behave like stem cells as they gain the properties of self renewal that is they can divide to give more cancer cells.Yamanaka factors are stem cell markers, proved by two scientists, Takahashi and Yamanaka that these transcription factors (then) were responsible for inducing pluoripotency in mouse (Hu et al 2010).Western Blot analysis of these factors will help us reconfirm that cell lines transfected with mutant p53 constructs, show stemness.

G) Side populations- Side Population Assay helps in detecting cancer cell stemness as this assay detects the efflux of dyes like Hoechst 33342 and PI suggesting the over-expression of ABC transporter proteins, which is a hallmark feature in stem cells. Viable cells (Side Population) that show Hoechst 33342 dye efflux could be identified to show stem characters. This is also an evidence to show how cancer cells with stem characters render themselves chemoresistant. In the graph obtained after performing FACS, the side population can be identified as few dots seen at the ‘tail’region at the left bottom corner (Hu et al 2010).

H) Chemoresistance can be detected by MTT assay: Chemoresistance is a characteristic which is associated with p53 Gain-of-Function mutations. MTT assay is a colorimetric assay which helps in checking for viability of cells. Cells that are metabolically active will form NADP(H) in the process of mitochondrial respiration, which can reduce MTT to form a compound called Formazan.

METHODOLOGY

We have worked with both cell lines and clinical samples simultaneously. Firstly we have taken p53 null cell line SKOV-3 and transiently transfected them with pCMV-Neo-Bam p53 R175H and pCMV-Neo-Bam p53 R273H constructs to check for the expression of mutant p53. Our next aim was to check for stemness in cancer cells, which we have done by performing two experiments- I) We have checked for expression of Yamanaka factors like Oct-4, c-Myc by performing Western blot, II)We have performed side population assay to check for stem properties of cancer cells. Besides, we also wanted to check for chemoresistance in cancer cells by MTT assay. Parallelly, we prepared blocks of normal and tumor tissue samples obtained from patients after surgery. These blocks were cut into 5-8 micron sections for downstream H&E staining and Immunohistochemistry. Immunohistochemistry was performed to check for the expression of p53 protein in the nucleus of the tissue section. We have also initiated the development of stable cell lines by transfecting SKOV3 cells with R175H and R273H constructs and empty vector.

Principles and Procedures

 I) Testing whether null cell line (SKOV-3) is actually null

Principle:

SKOV-3 cell line is a cancer cell line derived from ascites of a 64 year old caucasian woman with an ovarian serous cystadenocarcinoma. SKOV-3 cells are devoid of p53 protein as the RNA for p53 protein degrades due to Nonsense Mediated Decay. In contrast, A2780 cell lines have WT p53 which canonically undergoes MDM-2 mediated proteasomal degradation. This is why its expression cannot be easily detected. For this very reason, we treat both the cell lines with 5 Fluorouracil (5-FU), a DNA damaging agent which will trigger more p53 proteins to get activated. As SKOV-3 cells do not have WT p53 in them, actions of 5-FU does not effect the expression of p53 but in case of A2780 cell lines, 5-FU treatment induces robust expression of p53 protein, which can then be easily detected by Western Blotting.

Procedure:

 A) p53 null cell line (SKOV-3) and WT p53 cell line (A2780) were grown in two 35 mm dishes each.

B) One dish of each line was treated with DMSO and the other dish with 5-FU.

C)After 48 hours of 5-FU treatment, p53 protein expression was checked by Western Blotting.

II) Checking whether the pCMV-Neo-Bam plasmid contains mutant p53 DNA inserts

Principle:

ThepCMV-Neo-Bam vector comprises of two transcriptional units- I) a CMV promoter/enhancer upstream of multiple cloning site and II) a herpes simplex virus (HSV) thymidine kinase promoter/ enhancer upstream of the Neomycin resistant gene. pCMV- Neo-Bam p53 R175H and pCMV-Neo-Bam p53 R273H constructs were digested with Restriction Endonuclease, Bam HI and electrophoresed to check presence of the p53 insert. pCMV-Neo-Bam vector has a single Bam HI site whereas the above mentioned p53 mutant vectors have two Bam HI cut sites (Table 1). When these p53 mutant vectors digested with Bam HI are run in Agarose gel, two bands are formed at around 1.3 Kb (small fragment) and 6.5 Kb (large fragment) which clearly signifies that pCMV-Neo-Bam vector has mutant p53 DNA inserts.

PlasmidsBam HI cut sitepossible fragment sizes after BamHI digestion
pCMV-Neo-Bam p53 1329 
pCMV-Neo-Bam p53 R175H1329 , 2675 7900 (single cut), 6554, 1346 (2675-1329=1346)
pCMV-Neo-Bam p53 R273H1416, 23787824 (single cut), 6502, 1322 (2378-1416= 1322)

  Table 1: different product length after restriction digestion with BamHI

Procedure:

A) Concentration and purity of the plasmid DNA (previously prepared by plasmid midiprep Kit (Qiagen))was checked using NanoDrop (NanoDrop Lite Spectrophotometer, Thermo Fisher Scientific). 

B) 20µl reaction was set with 300ng DNA and 4µl of BamHI (20U/µl; New England Biolabs) in 10X NEB Buffer 3 at 37°C for 2 hours.  

C) Plasmids treated and untreated with Bam HI were run in the Agarose Gel.

D) After electrophoresis, plasmid bands on the the agarose gel was analysed.

III) Transient transfection of SKOV-3 cell line with R175H and R273H plasmids

Principle:

Transient transfection is the transfection of plasmids into cells wherein the plasmids enter the nuclei of the target cells but donot get incorporated in the host cell genome. As a result of which the expression of the gene in the plasmid remains for a short period of time (48 to 72 hours). In this experiment, we transfected the pre confirmed p53 null cell line SKOV-3 with p53 mutants R175H and R273H plasmids, separately by using Liofectamine 2000 (Thermo Fisher), and checked for the expression of mutant p53 by Western Blotting.

After transfecting the cells with plasmids, Western Blot is performed to check for incorporation of empty vector and p53 mutant plasmid in SKOV-3 cell lines. Firstly, a protein lysate from these cells is prepared for further separation by SDS-PAGE and analysis by Western Blotting (as described in a following section). We used antibodies against ptoteins like p53, B-actin,c-Myc and Oct-4, to detect the presence of these proteins.

Procedure:

A) Cells were starved for 1 hour in RPMI (-/-) media[-/-media is devoidof antibiotic and FBS]. 

B) 4 reactions were prepared in RPMI(-/-) media Untransfected ,Empty vector, p53 mutant R175H plasmid, p53 mutant R273H plasmid (Waited for 5mins) 

C) A mixture of lipofectamine 2000 (ThermoFisher) and RPMI (-/-)media was prepared. (Waited for 5 mins). 

D) I and II were mixed (waited for 20 mins). 

E) This mixture was added to starved cells and kept in incubator. 

F) After 4-6hrs, cells were given media change with RPMI (+/+) media[+/+ media contains both antibiotic and FBS].  

G) After 48 hrs, p53 expression was checked by Western Blot.

IV) Checking for cells exhibiting stemness by side population analysis

Principle:

Side population assay helps to identify the differential potential of cells to efflux Hoechst 33342. Stem cells exhibit the property of dye efflux through ABC transporters representing a specific population (Side population) of cells distinguishable from the Hoechst stained cells. When all the cells are incubated in Hoechst 33342 dye, cell take up this fluorescent dye as they have high affinity to bind to AT regions of DNA and can permeate into the cells without disturbing the cell membrane integrity. Thus Hoechst 33342 dye is used for live staining of cells. On the other hand, Propidium iodide (PI) stains the dead cells and can only reach the DNA when the cell membrane is disrupted. PI stain is used to distinguish between live and dead cells. Few cells in the population exhibits stem characters as a result of which has their ABC Transporters highly expressed. These cells take up Hoechst 33342 dye and efflux it out of the cells using ABC transporters. These specific group of cells are exhibited as the Side Population.

 Procedure:

A) SKOV-3 cells were transfected with pCMV-Neo-Bam R175H plasmids of different plasmid concentration (100ng, 250ng,500ng). 

B) After 48hrs, cells were harvested and 106 cells were resuspended in 1ml of RPMI (-/-) media supplemented with 2% FBS and addition of 5µg/ml Hoechst 3342 dye was done.

C) This mixture was incubated in water bath at 37℃ with intermittent shaking for 90 mins.

D) After incubation, the samples were spun to discard the supernatant which contains the dye.

E) Samples were washed with 1X PBS (ice cold). Pellets formed were resuspended in 500ul of ice cold 1X PBS and 2mg/ml PI was added and immediately readings were taken in Flow cytometer.

 V) Isolation of protein and checking protein expression btWestern Blotting

 Principle:

Western Blotting is a molecular biology technique which involves in transfer of proteins seperated by SDS-PAGE, onto a PVDF or Nitrocellulose membrane for further analysis with antibodies. For this, first aprotein lysate is required to be made. NP 40,which is a commercially available detergentis added to the cells transfected with p53 mutant, will break open the cell membrane to release the proteins in the lysate. Proteinase inhibitors are also used to block proteinase activity preventing protein degradation. After the protein lysate is prepared, the concentration of protein is checked spectrophotometrically at 595nm by performing Bradford Assay. The amount of protein to be seperated is calculated to finally load it onto the wells.

SDS-PAGE is a technique which seperates protein on the basis of their size, molecular weight and charge. SDS (Sodium Dodecyl Sulphate) is a detergent which imparts negative charge to all proteins that will run across the Polyacrylamide gel and denatures the complex structured proteins to their primary conformation, which help the proteins pass easily through the pores in the polyacrylamide gel.

After the proteins are seperated in the polyacrylamide gel, the bands are then transferred to a PVDF membrane by western blotting. A gel-membrane sandwich is prepared which is placed into the transfer buffer and subjected to voltage and current. The bands formed in the polyacrylamide gel gets transferred to the PVDF membrane. Further, the membranes are treated with blocking agents (5% BSA) which prevents non-specific binding of primary antibodies. Subsequently, primary antibodies and the HRP-conjugated secondary antibodies are added, with intermittent washing steps to reduce non-specific binding of these both. Primary antibodies attach to the specific proteins in the membrane and further secondary antibodies bind to the primary antibodies. Horse Raddish Peroxidase (HRP), that is conjugated with secondary antibodies, react with substrate luminol in the presence of Hydrogen Peroxide (H2O2), to emit light. This light is captured in a X-Ray film which is then developed to visualize the protein bands.

 Procedure:

A) The cells werelysedin NP-40buffer added with proteinase inhibitor by intermittent vortexing and incubation on ice, followed by centrifugation at 13000 rpm for 8mins at 4℃.

B) The protein lysate was taken and the concentration of protein present was measured spectrophotometrically by Bradford Assay. 

C) Accordingly, amount of protein to be loaded in the wells for SDS-PAGE run was calculated and the appropriate amount was loaded in the wells. 

D) The proteins were seperated in the polyacrylamide gel and bands were obtained. These proteins were then blotted on to PVDF membrane for checking the expression of Oct-4 (mouse monoclonal, abcam), c-Myc (rabbit monoclonal, abcam), p53 (mouse monoclonal, abcam) and B-actin (rabbit monoclonal, abcam) by the technique of Western Blotting.

E) After the proteins were blotted onto the PVDF membrane, these were developed in X ray films for further visualisation.

 VI) Testing drug resistance of cancer cells using MTT Assay

Principle:

MTT assay is a qualitative and colorimetric assay which helps in detecting viability of cells and their metabolic activities. MTT is a yellow colored compound, that is reduced by the action of NAD(P)H dependent oxidoreductase enzyme that is produced during mitochondrial respiration, to an insoluable purple coloured compound called Formazan. When cells are alive and metabolically active they tend to form more of NAD(P)H which inturn reduces more MTT into Formazan which could be detected by the change in colour. In case of drug resistance, cells that possess resistance towards drug will not die and yet produce NADPH which will reduce MTT into Formazan and accordinly colour change can be seen but those that are susceptible to drugs will die and not produce any NADPH to reduce MTT to Formazan. The colour change is analysed spectrophotometrically to get the absorbance values for each sample with different drug doses.

Procedure:

 A) Cells were seeded in each well of the 96 well plate for further transfection.

 B) After 24 hrs cells were transfected with 500ng of Empty vector, p53 R175H mutant vector and p53 R273H mutant vector each, in triplicates. 

C) After 24 hrs, 5-FU(drug) was added with a dosage of 0uM, 15uM, 30uM, 45uM, 60uM, 75uM, 90uM, 100uM.  

D) After 48 hrs, MTT was added to each well (in dark conditions as MTT is light sensitive) and kept in 37℃ for 3 hrs. 

E) After 3 hrs, DMSO was added to solubilise the insoluable Formazan which gives puple coloration. 

F) Spectrophotometric reading was taken at 595 nm.

VII) Block Preparation from Tumor and Normal tissues collected from HGSOC patients

 Principle:

Tissue samples collected from patients are difficult to be sectioned for further experiments. Thus, tissue samples go through several steps of dehydration with increasing concentrations of alcohol followed by preparation of blocks of paraffin in which the tissue is surrounded with paraffin in all its side. This helps for sectioning the tissues into very thin sections of approximately 5-8 microns which can be further placed on slides for Haematoxylin and Eosin (H&E) staining and immunohistochemistry (IHC).

 Procedure:

 Day I

A) The tissues were kept in 30ml glass bottle and kept under running tap water [keep for 1hour]

B) The tissue were transferred in 50% alcohol [kept for 2hours] 

C) The tissue were transferred in 70% alcohol [kept for overnight]

 Day II

D) Tissues were transferred in 90% alcohol [kept for 2 hrs]

E) Then kept at absolute alcohol for 2 hrs.

F) These were again put into xylene [Keep for 5 mins until the edge of tissue becomes translucent]

G) Tissues were put into preheated xylene:paraffin (1:1) [kept for 20mins at 65℃]

H) The tissues were then transferred to molten paraffin [Kept for 2 hrs at 65℃]

I) Blocks were prepared using L-blocks, with glycerol rubbed well on the inner walls of L-blocks before pouring in the molten paraffin. 

J) Before dismanteling the L-blocks, we waited for few minutes for the blocks to get solidified well.

VIII) Checking for histology of the cancer cells by H&E staining

Principle:

Hematoxylin and eosin (H&E) staining is one of the gold standard for checking the histology of a tissue. A tissue collected from patients suspected with cancerfor further biopsy, first undergoes H&E staining. H&E staining shows the general layout and distribution of cells and provides an overview of a tissue sample's structure. Hence a pathologist can easily differentiate between the nuclear and cytoplasmic parts of a cell. The hematoxylin stains cell nuclei blue, and eosin stains the extracellular matrix and cytoplasm pink, with other structures taking on different shades, hues, and combinations of these colors.

Procedure:

A) The tissue sections were deparaffinised by dipping the slides in xylene (twice, each for 10 mins).

B) Tissue sections wererehydratedin absolute alcoholby dipping the slide for 7mins.

C) Slides were dipped in 90% alcohol for 2 mins.

D) Slides dipped in 70% alcohol for2 mins. 

E) Slides werebrieflywashed in distilled water.

F) Slides containingtissue sections were dippedinMeyer’s hematoxylinsolutionand kept for 12 mins.

G) Tissue sectionsfloodedwith 1% acid-alcohol.

H) Tissues were dipped in warmtap waterand kep for 10 mins.

I) Slides were rinsed in distilled water.

J) Slides were dipped into 90% alcohol 10 times.

K) Tissue sections were counterstained in Eosin Ysolution for 40 seconds.

L) Tissue sections were subsequently dehydrated in 90%alcoholand100% alcohol(each for 5 mins).

M) The slides were dried and mounted using DPX, for further visualization.

IX) Checking for expression of p53 protein in tissue sections of clinical samples collected from HGSOC patients by immunohistochemistry

Principle:

Immunohistochemistry is a technique which helps us to see protein expression in the cells of a tissue section. In our experiment we wanted to check for the expression of p53 protein which should be ideally present in the nucleus of the cells. The protein antigens bind to the primary antibodies and these inturn bind to the secondary antibodies which are conjugated with Horse Raddish Peroxidase (HRP) enzymes. HRP uses DAB as the substrate and in presence of H2O2 show brown coloration, which implies the expression of the proteins in the cells.

 Procedure:

A) Tissue sections embedded on Poly-L lysine coated slides were kept in 65℃ [for20mins] 

B) Slides were transferred to xylene [kept for 10mins] 

C) Slides were transferred to another change of xylene [kept for 10mins] 

D) Slides were transferred to absolute alcohol [keep for 7 mins] 

E) Slides were transferred to 90% alcohol [keep for 5mins] 

F) Slides were transferred to 70% alcohol [keep for 5mins]

G) Slides were transferred to 1X PBS [twice, each for 5mins]

H) Slides were kept in boiling Tris EDTA (pH=9) under 1 psi pressure for 3 mins in autoclave/ pressure cooker. 

I) Slides were transfered to cold 1X PBS [for 5 mins]  

J) The tissue sections were flooded with 0.3% H2O2 [Kept for 7 mins]. Slides were washed in 1X PBS [twice, each for 5mins] 

K) Primary antibodies were added drop wise on the tissue sections and incubated overnight at 4°C 

L) Primary antibodies were washed off with 1X PBS [thrice, each for 5mins]and secondary antibodies were added on tissue sections [kept for 1 hr]

M) Secondary antibodies were washed off with 1X PBS [thrice, each for 5 mins]

N) A mixture of 0.5mg/ml DAB and 0.015% H2O2 in PBS was poured on the tissue sectionsand incubated for7 minutes 

O) Slides were dipped in Hematoxilin [Kept for 40secs] 

P) 1% acid-alcohol was prepared and added dropwise on tissue section [kept for 1-].

Q) Slide were dipped in tap water, enhanced blueing [kept for 3mins] 

R) Slides were dipped in 70% alcohol

S) Slides were dipped in 90% alcohol

T) Slides were transferred to 100% alcohol for 5 mins. 

W) Slides were dried and mounted with DPX for visualizing under the microscope.

XPreparation of SKOV-3 stable cell line

 Principle:

Stable transfection unlike transient transfection is the process in which the transfected DNA in the host cell is integrated in the host genome. The introduced DNA that gets incorporated in the genome will get passed onto their progeny

 Procedure:

 A) Transfection of cells using RPMI (-/-) media[-/-media is devoid of FBS and antibiotics] 

 B) Media change with RPMI (+/+) media [+/+ media contains both FBS and antibiotic.

C) Selection with G418 antibiotic(Neomycin)

D) Ongoing maintenance under selection.

RESULTS

 I) To test whether p53 null cell line (SKOV-3) is actually null

 A2780 cell lysate treated with 5-FU shows p53 expressions. SKOV-3 cell lysate and SKOV-3 cell lysate treated with 5 FU donot show any p53 expression which indicates that SKOV-3 cell lines donot contain p53 proteins.

 II) To check whether the pCMV-Neo-Bam plasmid contains mutant p53 DNA inserts

 p53 mutant R175H and R273H vector were digested with Bam HI and run on agarose gel. Two bands were obtained around 1.3Kb and 6.5Kb which signifies that pCMV-Neo-Bam has p53 mutant R175H and R273H DNA inserts.  

 III) Transient transfection of SKOV-3 cell line with R175H and R273H plasmids

p53 bands were seen in cells transiently transfected with p53 R175H mutant vector and p53 R273H mutant vectors. With the increase in dose of vector from 500 ng to 1 µg, band intensity also increased which implies p53 expression also increased. B-actin bands were seen in all the lanes whereas p53 bands were not seen in the lane of untransfected and empty vectors.

 IV) To check for cells exhibiting stemness by side population analysis

In untransfected cells, unstained with PI, cells are seen to take up Hoechst 33342 dye. In untransfected cells stained with PI, the average cell count in SP was 2 where in cells transfected with 100 ng R175H mutant vectors, average cell count was seen to be around 53. The average cell count for cells transfected with 150 ng and 500 ng of p53 mutant R175H construct was 43 and 34 respectively. Although all cells took up PI stain which implies that cells were dying, the cell count in side population increased from untransfected to transfected cells. It is under investigation process, as to why all the cells were taking up PI stain, but this increase in cell number in transfected in comparison to untransfected cells may indicate that stemness is related with mutant p53 expression.

V) To check for expression of stem cell markers by Western Blotting

c-Myc and Oct- 4 (faintly) band intensity increased from transfection with 100ng to 250 ng of R175H plasmid.which implies stem characters may be induced in cell line transfected with p53 R175H mutant.  

 VI)  To check for drug resistance of cancer cells by MTT assay

In untransfected and empty vector-transfected cells,decreasing trend of viability was seen with increase of drug dosage. In R175H transfected cells, no such decrease in trend was seen. In R273H, there was a decreasing trend but few intermittent doses failed to show effect. This may have indicated chemoresistance.

VII) Preparation of blocks of Normal and Tumor tissues collected from patient samples

After the tissue undergoes the various procedures, it is finally paraffinized which makes it easier to cut thin sections of the tissues (5-8 microns) for further H&E and IHC. Block preparation also prevents microbial activity in tissues which helps in storage.

VIII) Checking for histology of tumor tissue from patients by H&E staining

After performing H&E staining of tissues from patients, we see that the cell nuclei take up hematoxylin and stain blue while cytoplasm stain pink as it takes up eosin. In cancer tissues stained with hematoxylin and eosin, the nuclei often seem to be enlarged implying enhanced DNA replication.

The nuclei to cytoplasmic ratio is seen to be more.

IX) Performing Immunohistochemistry in clinical tissues to find out expression of p53 proteins

p53 protein expressions were seen in nuclei of the cells in the tissue section which is demonstrated by the presence of brown colour because of DAB staining. The staining is undergoing further standardization.

X) Preparation of stable SKOV-3 cell lines

The cells are under on going maintenance under selection with G-418 and this process usually takes time.

DISCUSSIONS

Our main motive of performing these experiments were to find out a relation between p53 Gain-Of-Function mutations with stemness and chemoresistance. Firstly we check for the p53 null cell lines and the p53 mutant DNA inserts in pCMV-Neo-Bam vector. These SKOV-3 cell lines were transiently transfected with p53 mutant R175H vectors to check for the p53 expressions. p53 protein expressions were checked by performing Western Blot which showed p53 protein bands clearly at 53 kDa.

Further experiments were performed for assessing stem characters. Western Blotting was performed to detect expression of Oct-4 and c-Myc which are also called Yamanaka factors, which are found to induce pluripotency in differentiated cells. In this experiment, the resolution of bands were faint and also the X Ray films deteriorated with time as a result of which the picture taken is not very clear. The amount of DNA that was loaded in the wells should have been more (around 70 μg) for a better result, but we are standardizing as to how we could load more lysate in the wells. Stemness was also checked by performing Side Population Assay, in which all our cells took up PI, which should not be the actual case. We are investigating about this as to why all the cells took up PI stain. In this experiment, a substantial increase in cell count in transfected samples were seen as compared to untransfected samples, which may imply that number of Side Population cells may alter with transfection of R175H mutant vectors. We checked for chemoresistance by performing a qualitative MTT assay which showed indications of chemoresistance. However, these experiments are required to be repeated before confirmation. We also standardized p53 IHC on clinical tissues samples. p53 expressions were seen in the nucleus of the tissues.

CONCLUSION

We could detect p53 expressions clearly in cells transfected with mutant p53 vectors. The relation of p53 GOF with stemness was seen upto certain extent but the experiments are in the standardization process. In the case of MTT assay, chemoresistance was qualitatively analysed but until it is quantitatively analysed we cannot comment strongly on the point. Experiments with clinical samples like IHC also is in the standardization process for getting better stained view under microscope. In previous researches in case of other cancers, there is already a conclusion drawn which showed p53 GOF mutations has relation with stemness and also chemoresistance is a result of Gain-of-Function mutations. This project contributed mainly in standardization of the experiments required to study the broad objective of finding out the role of GOF mutant p53 in stemness-associated chemoresistance in HGSOC. 

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